There are two related projects in this Program. Project 1. Epigenetic control of hematopoiesis - DNA methylation. In this project we will define the methylation profile of erythroid, and megakaryocytic, cells as well as their progenitors and other lineages to determine the DNA methylation status of regulatory elements associated with lineage choice. These data will be correlated with both mRNA expression profiles and the chromatin architecture studies in Project 2. Project 2. Epigenetic control of hematopoiesis histone modification and chromatin structure. In this project we will focus on the chromatin landscape of the genome in the same cells as in Project 1. We will determine the chromatin accessibility as well as the methylation and acetylation of histone proteins across the genome and correlated these with mRNA expression and DNA methylation. All of the cells in the peripheral blood are generated from a small population of hematopoietic stem cells (HSC) through a process of proliferation and differentiation known as hematopoiesis. The hematopoietic differentiation program includes well-defined stages at which the progeny of HSC become restricted to specific fates. The goal of this project is to define specific molecular signatures associated with specific stages of hematopoiesis. Project 1. Epigenetic control of hematopoiesis - methylation. Epigenetic changes, including DNA methylation, are an essential element of normal development and hematopoietic differentiation. Our goal is to comprehensively map these changes in the genomes of primary mouse erythroblasts, megakaryocytes and their immediate precursors, the Colony Forming Unit Erythroid (BFU-E) and Megakaryocyte Colony Forming Unit (CFU-MK). We will also profile more primitive progenitor cells including the Common Myeloid Progenitor (CMP) which gives rise to erythroid megakaryocytic, monocytic, and granulocytic lineages and HSC. The ENCODE project has given us a roadmap of the epigenetic landscape of different cell lines, but to move ENCODE further, we have decided to focus on primary mouse hematopoietic cells isolated from bone marrow. Following up on our initial analysis of genome-wide methylation (Hogart A, Lichtenberg J, Ajay SS, Anderson S, NIH Intramural Sequencing Center, Margulies EH, Bodine DM. Genome-wide DNA methylation profiles in hematopoietic stem and progenitor cells reveal overrepresentation of ETS transcription factor binding sites. Genome Res. 22 (8) 1407-18, 2012), we have now shown that while methylation decreases as erythroid cells mature, increased methylation is associated with differentiation in the megakaryocyte and granulocyte lineages. We have also generated RNASeq transcriptome profiles of both mRNA coding as well as non-coding RNAS in these cells (Paralkar VR, Mishra T, Luan J, Yao Y, Kossenkov AV, Anderson SM, Dunagin M, Pimkin M, Gore M, Sun D, Konuthula N, Raj A, An X, Mohandas N, Bodine DM, Hardison RC, Weiss MJ. Lineage and species-specific long noncoding RNAs during erythro-megakaryocytic development. Blood. 123(12):1927-37, 2014). Finally, we have developed new software packages to analyze these data that are becoming widely used in the field. (Lichtenberg J, Hogart A, Battle S. Bodine DM. Discovery of motif-based regulatory signatures in whole genome methylation experiments. Bioinformatics Open Source Conference (BOSC), 2012, Long Beach USA). We have found that while DNA methylation in promoter regions is negatively correlated with mRNA transcription, DNA methylation in the bodies of genes is positively correlated with mRNA transcription. This scenario is reversed for non-coding RNAs. De novo DNA methylation is a prominent feature of megakaryocyte and granulocyte/monocyte differentiation. However, the DNA methylation pattern in erythroblasts is a subset of what is present in HSC and CMP, demonstrating epigenetic memory. Project 2. Epigenetic control of hematopoiesis transcription factor occupancy. We have entered into a consortium with a group at Penn State led by Ross Hardison to take advantage our respective labs' strengths. The consortium is known as VISION (Validated Integrated Systematic InterpretatiON of hematopoietic epigenomes). In addition to the DNA methylation profiles described above, our group is generating histone methylation and acetylation profiles for erythroblasts, megakaryocytes their immediate precursors, GMP, CMP and HSC. We have provided the Hardison lab with cells RNASeq analysis of the transcriptome and ATACSeq for analysis of chromatin accessibility. We have completed ATACSeq, RNASeq and ChIPSeq analyses of 5different histone modifications in erythroblasts, megakaryocytes and CMP. Our findings include the identification of over 600 novel long non-coding RNAs, many of which are expressed specifically in one of the three cell types. We have identifies a set of enhancers defined by their histone code. The most active of these enhancers are defined as super enhancers and these have been shown to be entirely lineage specific. Consistent with our methylation data, the erythroid super enhancers are present in CMP (but not necessarily correlated with gene expression) while the megakaryocyte super enhancers arise de novo. We plan to extend these analyses by correlating the changes in gene expression we observe with the changes in the epigenetic profile of the genome to allow for the first time direct comparisons of gene expression and epigenetic signatures in primary cells.